In-depth circulating tumor DNA sequencing for prognostication and monitoring in natural killer/T-cell lymphomas.

Background: Epstein-Barr virus (EBV) quantitation and current imaging modalities are used for diagnosis and disease monitoring in Extranodal NK/T cell lymphoma (ENKTL) but have limitations. Thus, we explored the utility of circulating tumor DNA (ctDNA) as a diagnostic biomarker. Methods: Through in-depth sequencing of 118 blood samples collected longitudinally at different time points from 45 patients, we examined the mutational profile of each sample, estimated its impact on the clinical outcome, and assessed its role as a biomarker in comparison with EBV DNA quantitation. Results: The ctDNA concentration was correlated with treatment response, stage, and EBV DNA quantitation. The detection rate of ctDNA mutation was 54.5%, with BCOR (21%) being the most commonly mutated gene in newly diagnosed patients; TP53 mutation (33%) was the most prevalent in patients that experienced a relapse. Additionally, patients in complete remission exhibited a rapid clearance of ENKTL-related somatic mutations, while relapsed patients frequently presented with persisting or emerging mutations. We detected ctDNA mutations in EBV-negative patients (50%) and mutation clearance in EBV-positive patients in remission, suggesting ctDNA genotyping as an efficient complementary monitoring method for ENKTL. Additionally, mutated DDX3X (PFS HR, 8.26) in initial samples predicted poor outcome. Conclusion: Our results suggest that ctDNA analysis can be used to genotype at diagnosis and estimate the tumor burden in patients with ENKTL. Furthermore, ctDNA dynamics indicate the potential use of testing it to monitor therapeutic responses and develop new biomarkers for precision ENKTL therapy.

Recurrent somatic mutations and low germline predisposition mutations in Korean ALL patients.

In addition to somatic mutations, germline genetic predisposition to hematologic malignancies is currently emerging as an area attracting high research interest. In this study, we investigated genetic alterations in Korean acute lymphoblastic leukemia/lymphoma (ALL) patients using targeted gene panel sequencing. To this end, a gene panel consisting of 81 genes that are known to be associated with 23 predisposition syndromes was investigated. In addition to sequence variants, gene-level copy number variations (CNVs) were investigated as well. We identified 197 somatic sequence variants and 223 somatic CNVs. The IKZF1 alteration was found to have an adverse effect on overall survival (OS) and relapse-free survival (RFS) in childhood ALL. We found recurrent somatic alterations in Korean ALL patients similar to previous studies on both prevalence and prognostic impact. Six patients were found to be carriers of variants in six genes associated with primary immunodeficiency disorder (PID). Of the 81 genes associated with 23 predisposition syndromes, this study found only one predisposition germline mutation (TP53) (1.1%). Altogether, our study demonstrated a low probability of germline mutation predisposition to ALL in Korean ALL patients.

Comprehensive Laboratory Analysis of Korean Acute Alcoholic Intoxication Patients Reveals the Need for a National Hepatitis B Virus Vaccination Program in Korea.

BACKGROUND: Acute alcoholic intoxication patients (AAIP) are a common public health problem. The aim of this study was to perform a comprehensive laboratory analysis for these patients to investigate the co-morbid medical problem. METHODS: We retrospectively reviewed laboratory findings of AAIP who were transferred to the emergency department (ED) from January 2017 to June 2017. RESULTS: A total of 160 male patients were enrolled. Sixteen patients (16/160, 10.0%) and three patients (3/160, 1.9%) had macrocytic anemia and microcytic anemia, respectively. A total of 33 patients (33/160, 20.6%) showed thrombocytopenia (<150×109 /L). Twelve patients (12/159, 7.5%) showed low serum albumin level (<3.5 g/dL). Three patients (3/160, 1.9%) had chronic kidney disease stages 3-4 based on estimated glomerular filtration rate. Six patients (6/27, 22.2%) had high hemoglobin A1c (HbA1c) level (>7.0%). Positive rates of hepatitis B surface antigen and antiHBs antibody (anti-HBs Ab) were 3.5% (5/141) and 49.0% (68/141), respectively. CONCLUSION: Patients with AAIP who were transferred to ED had various laboratory abnormalities (anemia, thrombocytopenia, high HbA1c). They had low positive rate of anti-HBs Ab. This might be a public health problem, suggesting the need of hepatitis B virus vaccination program for AAIP. Our data suggest the need of further nationwide studies.

Hb Heathrow [ß103(G5)Phe→Leu], a First Report in an Asian Patient with Erythrocytosis.

Congenital erythrocytosis (CE) is a rare and heterogeneous disease. The high oxygen affinity hemoglobin (Hb) variants are the most common cause of CE. Herein, we report a Korean patient with isolated erythrocytosis. A 25-year-old man was referred to our hospital for evaluation of high Hb level (Hb 20.4 g/dL, hematocrit 58%, reticulocyte count 2.90%, white blood cell count 6.83×109/L, and platelet count 195×109/L). Bone marrow biopsy revealed normocellular marrow without myeloproliferative features. JAK2 (V617F, exon 12), CALR (exon 9), and MPL W515K/L mutations were not detected. P50 (partial pressure at which Hb is half saturated with oxygen), which is an indicator of left-shift of oxygen dissociation curve (high oxygen affinity state), was 14.3 mm Hg (reference value 22.6-29.4 mm Hg). He was suspected to have CE. Mutation analysis of the HBB gene revealed the known Hb variant, Hb Heathrow [ß103(G5)Phe→Leu]. This is the first report of Hb Heathrow in Asian.

Prognostic Implications of Monosomies in Patients With Multiple Myeloma.

BACKGROUND: Cytogenetic analysis aides in risk stratification for patients with multiple myeloma (MM). Although several cytogenetic aberrations have been reported to be prognostic, less is known about the association between the presence of monosomies and prognosis. The present study evaluated the prevalence and prognostic implications of monosomies in patients with MM. MATERIALS AND METHODS: Karyotypes were determined using conventional cytogenetics and fluorescence in situ hybridization (FISH). The prognostic effect of monosomies was evaluated by comparison with the clinical factors in MM patients with normal karyotypes. RESULTS: Karyotypes were successfully determined in 167 of the 170 patients with MM. Of these 167 patients, 52 (31.1%) had abnormal karyotypes. Univariable analyses showed that a normal karyotype, hypodiploidy, monosomies of chromosomes 13 and 16, deletion or monosomy of 13q14, and loss of X detected by metaphase analysis were each associated with reduced progression-free survival (P < .05 for each). Univariable analyses showed that a normal karyotype, hypodiploidy, monosomies of chromosomes 13 and 16, deletion or monosomy of 13q14 detected by metaphase analysis and FISH-determined RB1 (13q)/TP53 (17p) deletion were each associated with reduced overall survival (P < .05 for each). Multivariable analysis showed that hypodiploidy detected by metaphase analysis was independently prognostic of shorter progression-free survival (P < .05 for each) and that hypodiploidy, monosomy 16, and loss of Y chromosome and FISH-determined TP53 (17p) deletion were associated with reduced overall survival (P < .05 for each). CONCLUSION: In addition to known cytogenetic abnormalities, such as monosomy 13, hypodiploidy, and TP53 (17p) deletion, monosomy 16 and loss of the Y chromosome have adverse prognostic implications in patients with MM.

Detection of MYD88 L265P in patients with lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia and other B-cell non-Hodgkin lymphomas.

BACKGROUND: Recent studies have identified a high prevalence of the MYD88 L265P mutation in lymphoplasmacytic lymphoma (LPL)/Waldenstrom macroglobulinemia (WM) cases, whereas low frequencies have been observed in other B cell non-Hodgkin lymphomas (NHLs). METHODS: We evaluated the sensitivity of the mutant enrichment 3'-modified oligonucleotide (MEMO)-PCR technique, a new detection method. We examined the MYD88 L265P mutation in a series of Korean patients with LPL/WM and other B cell NHLs in bone marrow aspirates, using the MEMO-PCR technique. RESULTS: The sensitivity of MEMO-PCR was estimated to be approximately 10-16.7%. MYD88 L265P was detected in 21 of 28 LPL cases (75%) and only three of 69 B cell NHL cases (4.3%). CONCLUSION: Although MEMO-PCR had relatively low sensitivity, we confirmed the high prevalence of the MYD88 L265P mutation in Korean LPL patients. Our study suggests the diagnostic value of MYD88 L265P for differentiating B-cell NHLs.

Mutation profiling of 19 candidate genes in acute myeloid leukemia suggests significance of DNMT3A mutations.

We selected 19 significantly-mutated genes in AMLs, including FLT3, DNMT3A, NPM1, TET2, RUNX1, CEBPA, WT1, IDH1, IDH2, NRAS, ASXL1, SETD2, PTPN11, TP53, KIT, JAK2, KRAS, BRAF and CBL, and performed massively parallel sequencing for 114 patients with acute myeloid leukemias, mainly including those with normal karyotypes (CN-AML). More than 80% of patients had at least one mutation in the genes tested. DNMT3A mutation was significantly associated with adverse outcome in addition to conventional risk stratification such as the European LeukemiaNet (ELN) classification. We observed clinical usefulness of mutation testing on multiple target genes and the association with disease subgroups, clinical features and prognosis in AMLs.

Bone Marrow Suppression and Hemophagocytic Histiocytes Are Common Findings in Korean Severe Fever with Thrombocytopenia Syndrome Patients.

The causes of cytopenia in patients with severe fever with thrombocytopenia syndrome (SFTS) are not fully understood until now. We reviewed the bone marrow (BM) findings of patients with SFTS to unravel the cause of the cytopenia. Three Korean SFTS were enrolled in this study. Thrombocytopenia, neutropenia, and anemia were detected in all three patients. Severe hypocellular marrow (overall cellularity <5%) and a decreased number of megakaryocytes were noted in one patient, and hypo-/normocellular marrow and an increased number of hemophagocytic histiocytes were observed in two patients. Megakaryocytes were relatively preserved in two patients. Although a limited number of cases are available, our observations suggest that both BM suppression and peripheral destruction or sequestration are causes of cytopenia of patients with SFTS. To the best of our knowledge, this is the first well documented pathologic evaluation of Korean SFTS.

A Novel Hemoglobin Variant Associated with Congenital Erythrocytosis: Hb Seoul [ß86(F2)Ala→Thr] (HBB:c.259G>A).

We report the identification of a novel hemoglobin (Hb) variant [ß86(F2)Ala→Thr; HBB: c.259G>A], Hb Seoul, causing congenital erythrocytosis due to high oxygen affinity. The patient was a 33-year-old Korean man with isolated erythrocytosis. JAK2 somatic mutations were negative. Direct sequencing analyses revealed that the patient was heterozygous for c.259G>A, while other known causative genes (BPGM, EGLN1, EPAS, EPOR and VHL) had no mutation. ß86(F2) is a critical residue that affects the oxygen affinity. The novel variant in our patient, Hb Seoul, adds to the previously reported 4 other Hb variants from ß86(F2) substitutions that cause congenital erythrocytosis.

Usefulness of Flow Cytometric Analysis for Detecting Leptomeningeal Diseases in Non-Hodgkin Lymphoma.

BACKGROUND: The clinical usefulness of flow cytometry (FCM) for the diagnosis of leptomeningeal diseases (LMD) in non-Hodgkin lymphomas has been suggested in previous studies but needs to be further validated. With this regards, we evaluated the use of FCM for LMD in a series of Korean patients with non-Hodgkin lymphoma. METHODS: FCM and cytomorphology were conducted using samples obtained from clinically suspected LMD patients, follow-up LMD patients, and those with high risk of developing tumorigenic diseases. We then compared results of FCM and cytomorphology. In total, 55 and 47 CSF samples were analyzed by FCM and cytomorphology, respectively. RESULTS: Of the samples analyzed, 25.5% (14/55) and 12.8% (6/47) were positive by FCM and cytomorphology, respectively. No samples were determined as negative by FCM but positive by cytomorphology. Seven patients were positive only by FCM and negative by cytomorphology, and six among them were clinically confirmed to have LMD either by follow-up cytomorphology or imaging study. CONCLUSIONS: We observed a high detection rate of tumor cells by FCM compared with cytomorphology. FCM study can be useful in early sensitive detection of LMD.

Antigen Expression Patterns of Plasma Cell Myeloma: An Association of Cytogenetic Abnormality and International Staging System (ISS) for Myeloma.

BACKGROUND: Immunophenotyping of plasma cell has become an important diagnostic tool for plasma cell myeloma. There have been a few studies for association of antigen expression and cytogenetic abnormality of plasma cell myeloma. METHODS: A total of 68 symptomatic/smoldering plasma cell myeloma case were analyzed by multicolor flow cytometry using CD38 and CD138 for primary gating of plasma cells. A conventional cytogenetics and fluorescence in situ hybridization (FISH) studies for detection of del(13q) or aneuploidy, del(17p), and IGH/FGFR translocation were done. We statistically analyzed the association of antigen expression and cytogenetic abnormality/myeloma stage (international staging system for multiple myeloma). RESULTS: Positive expression of CD19, CD28, CD45, CD56, CD117, and CD274 was detected in 8.8%, 50.0%, 50.0%, 75.0%, 39.7%, and 2.9% of cases, respectively. CD117-negative cases were associated with hypodiploidy (P = 0.017). CD45-negative cases were associated with deletion 13 or aneuploidy (P < 0.001) and del(17p)(P = 0.011) by FISH. CD45-negativity or CD117-negativity was associated with advanced stage (P = 0.012 and P = 0.016, respectively). CONCLUSION: The antigen expression patterns of myeloma plasma cell were associated with cytogenetic abnormality and stage.

Mutant Enrichment with 3'-Modified Oligonucleotides (MEMO)-Quantitative PCR for Detection of NPM1 Mutations in Acute Myeloid Leukemia.

BACKGROUND: Detection of NPM1 mutations in acute myeloid leukemia (AML) is important for risk stratification, treatment decision, and therapeutic monitoring. We have designed a real-time PCR method implementing the Mutant enrichment with 3'-modified oligonucleotides (MEMO) technique to detect NPM1 mutations and validated its utility in clinical samples. METHODS: Sensitivity and linearity were evaluated using serially diluted NPM1-positive samples. Clinical usefulness was assessed by measuring the levels of mutant alleles in 29 patients at diagnosis and in ten patients after induction chemotherapy. RESULTS: Excellent linear relationships between the mutant allele proportion and the threshold cycle (Ct) values (r = 0.999) were observed in a range of 1:1-1:10(3) . MEMO-PCR was able to detect NPM1 mutations regardless of mutant type and also detected novel mutants (964_967delTGGAinsATGATGTC, 957_959delCTGinsATGCATG, 960insTAAG, and 960insTCAG). The concentrations of NPM1 mutant alleles decreased after induction chemotherapy in accordance with the reduction of tumor cells, and in one case, NPM1 mutant alleles were detectable about 7 months before morphological relapse. CONCLUSION: MEMO-quantitative PCR was shown to detect virtually all types of NPM1 mutants with high sensitivity and specificity. This novel method may be useful in the diagnosis of AML with an NPM1 mutation, the detection of minimal residual disease, and the monitoring of treatment response.